Glycoproteins are an important class of biomolecules that play crucial roles in many biological events such as cell adhesion, tumor metastasis, pathogen infection, and immune response. Most mammalian cell surface proteins and human serum proteins are glycoproteins and it is not surprising then that therapeutic glycoproteins are an important class of biotechnology products. These include, amongst many others, granulocyte macrophage-colony stimulating factor, tissue plasminogen activator, interleukin-2, erythropoietin (EPO), and antibodies. Both natural and recombinant glycoproteins are typically produced as a mixture of glycoforms that differ only in the structure of the pendent oligosaccharides. This heterogeneity in glycosylation is a major problem in structural and functional studies of glycoproteins (e.g. crystallization studies), as well as in development of glycoprotein drugs. The attached sugar chains may for instance have profound effects on protein folding, stability, action, pharmacokinetics, and serum half-life of the glycoprotein, and some sugar chains are very immunogenic.
Glycosylation is one of the most common post-translational modifications of proteins in eukaryotes. N-glycosylation is a highly conserved metabolic process, which in eukaryotes is essential for viability. Protein N-glycosylation originates in the endoplasmic reticulum (ER), where an N-linked oligosaccharide (Glc3Man9GlcNAc2) assembled on dolichol (a lipid carrier intermediate) is transferred to the appropriate asparagines residue (Asn) of a nascent protein. This is a co-translational event largely common to all eukaryotic organisms. The three glucose residues and one specific α-1,2-linked mannose residue are removed by specific glucosidases and an α-1,2-mannosidase in the ER, resulting in the core oligosaccharide structure, Man8GlcNAc2. Proteins with this core sugar structure are transported to the Golgi apparatus where the sugar moiety undergoes various modifications. Glycosyltransferases and mannosidases line the inner (luminal) surface of the ER and Golgi apparatus and thereby provide a catalytic surface that allows for the sequential processing of glycoproteins as they proceed through the ER and Golgi network. The multiple compartments of the cis, medial, and trans Golgi and the trans Golgi Network (TGN), provide the different localities in which the ordered sequence of glycosylation reactions can take place. As a glycoprotein proceeds from synthesis in the ER to full maturation in the late Golgi or TGN, it is sequentially exposed to different glycosidases, mannosidases and glycosyltransferases such that a specific N-glycan structure may be synthesized. There are significant differences in the modifications of the sugar chain in the Golgi apparatus between lower and higher eukaryotes.
In higher eukaryotes, the N-linked oligosaccharides are typically high mannose, complex and mixed (hybrid) types of structures that vary significantly from those produced in yeast (Kornfeld et al., Ann. Rev. Biochem. 54: 631-664 (1985)). In mammalian cells, the modification of the sugar chain can follow 3 different pathways depending on the protein moiety to which it is added. That is: (1) the core sugar chain does not change; (2) the core sugar chain is changed by adding the N-acetylglucosamine-1-phosphate moiety (GlcNAc-1-P) in UDP-N-acetyl glucosamine (UDP-GlcNAc) to the 6-position of mannose in the core sugar chain, followed by removal of the GlcNAc moiety to form an acidic sugar chain in the glycoprotein; and (3) the core sugar chain is first converted into Man5GlcNAc2 by removing 3 mannose residues with Golgi α-Mannosidase I; Man5GlcNAc2 is then further modified by adding GlcNAc and removing 2 more mannose residues, followed by sequentially adding GlcNAc, galactose (Gal), GalNAc, fucose and N-acetylneuraminic acid (also called sialic acid (NeuNAc)) to form various hybrid or complex sugar chains (R. Kornfeld and S. Kornfeld, 1985; Chiba et al., 1998). Different organisms provide different glycosylation enzymes (glycosyltransferases and glycosidases) and different glycosyl substrates, so that the final composition of a sugar side chain may vary markedly depending upon the higher eukaryotic host. Typically, the protein N glycans of animal glycoproteins have bi-, tri-, or tetra-antennary structures. These branched structures are synthesized by the GlcNAc transferase-catalyzed addition of GlcNAc to regions of the oligosaccharide residue. Subsequent to their formation, the antennary structures are terminated with different sugars including Gal, GalNAc, GlcNAc, fucose (Fuc) and sialic acid residues.
In yeast and filamentous fungi (lower eukaryotes), only a part of the Man8(9)GlcNAc2 structures are (partially) trimmed down to Man5GlcNAc2. These oligosaccharides can then be further modified to fungal-specific glycans through the addition of mannose and/or mannosephosphate residues in a diester linkage. The resulting glycans are known as “high-mannose” type glycans or mannans. For example, yeast glycopeptides include oligosaccharide structures that consist of a high mannose core of 9-13 mannose residues, or extended branched mannan outer chains consisting of up to 200 residues (Ballou, et al., Dev. Biol. 166: 363-379 (1992); Trimble et al., Glycobiology 2: 57-75 (1992)).
Considerable effort has been directed towards the identification and optimization of new strategies for the preparation of glycopeptides and glycoproteins for therapeutic application. Probably the most documented approach amongst the many promising methods is the engineering of cellular hosts that produce glycopeptides having a desired glycosylation pattern. For a recent review on how this can be achieved, in particular in yeast, see Wildt et al., Nature reviews 2005, 119-28; and Hamilton et al., Curr Opin Biotechnol. 2007; 18(5):387-92. Other exemplary methods include chemical synthesis, enzymatic synthesis, enzymatic remodeling of formed glycopeptides and of course methods that are hybrids or combinations of one or more of these techniques.
Regarding cell host systems, in principle, mammalian, insect, yeast, fungal, plant or prokaryotic cell culture systems can be used for production of most therapeutic and other glycopeptides in commercially feasible quantities. In practice, however, a desired glycosylation pattern on a recombinantly produced protein is difficult to achieve. For example, bacteria do not N-glycosylate via the dolichol pathway, and yeast only produces oligomannose-type N-glycans, which are not generally found in large quantities in humans. Similarly, plant cells do not produce sialylated oligosaccharides, a common constituent of human glycopeptides. In addition, plants add xylose and/or α-1,3-linked fucose to protein N-glycans, resulting in glycoproteins that differ in structure from animals and are immunogenic in mammals (Lerouge et al., Plant Mol Biol. 1998; 38(1-2):31-48; Betenbaugh et al., Curr Opin Struct Biol. 2004; 14(5): 601-6; Altmann, Int Arch Allergy Immunol. 2007; 142(2):99-115). As recently reviewed, none of the insect cell systems presently available for the production of recombinant mammalian glycopeptides will produce glycopeptides with the same glycans normally found when they are produced in mammals (Harrison and Jarvis, 2006, 159). Moreover, glycosylation patterns of recombinant glycopeptides may also differ when produced under different cell culture conditions (Watson et al. Biotechnol. Prog. 10: 39-44 (1994); and Gawlitzek et al., Biotechnol. J. 42: 117-131 (1995)) or even between glycopeptides produced under nominally identical cell culture conditions in two different bioreactors (Kunkel et al., Biotechnol. Prog. 2000: 462-470 (2000)).
Thus, despite significant advances in this field, heterogeneity of glycosylation remains an issue. Heterogeneity in the glycosylation of recombinantly produced glycopeptides arises because the cellular machinery (e.g., glycosyltransferases and glycosidases) may vary from species to species, cell to cell, or even from individual to individual. The substrates recognized by the various enzymes may be sufficiently different that glycosylation may not occur at some sites or may be vastly modified from that of the native protein. Glycosylation of recombinant proteins produced in heterologous eukaryotic hosts will often differ from the native protein. Therapeutic glycoproteins are typically produced in cell culture systems as a mixture of glycoforms that possess the same peptide backbone but differ in both the nature and site of glycosylation. The heterogeneity in glycosylation poses significant difficulty for the purification, efficacy, as well as therapeutic safety of glycoproteins. Cell and/or glyco-engineering and some biochemical modifications may have yielded cells or (e.g. yeast) strains that produce recombinant glycoproteins with predominant glycoforms but, in most cases, as with natively expressed glycoproteins, the structures that have been obtained remain heterogeneous. Notably, different glycosylation forms can exert significantly different effects on the properties of a given protein, and some glycoforms can even cause allergy problems and undesired immune responses. This is e.g. particularly true for the high-mannose-type glycoproteins normally produced in yeast. Isolation of a glycoprotein having a particular glycosylation state from such a mixture of glycosylation forms is extremely difficult. However, as small amounts of impurities can dramatically interfere with the desired activities of the glycoprotein of interest, such inhibition is also highly desirable.
In addition to preparing properly glycosylated glycopeptides by engineering the host cell to include the necessary compliment of enzymes, efforts have been directed to the development of both de novo synthesis of glycopeptides and the in vitro enzymatic methods of tailoring the glycosylation of glycopeptides. Although great advances have been made in recent years in both carbohydrate chemistry and the synthesis of glycopeptides (Arsequell et al., Tetrahedron: Asymmetry 10: 3045 (1999)), there are still substantial difficulties associated with chemical synthesis of glycopeptides, particularly with the formation of the ubiquitous β-1,2-cis-mannoside linkage found in mammalian oligosaccharides. Moreover, regio- and stereo-chemical obstacles must be resolved at each step of the de novo synthesis of a carbohydrate.
As enzyme-based syntheses have the advantages of regioselectivity and stereoselectivity, the use of enzymes to synthesize the carbohydrate portions of glycopeptides is a promising approach to preparing glycopeptides. Moreover, enzymatic syntheses can be performed using unprotected substrates. Three principal classes of enzymes are used in the synthesis of carbohydrates, glycosyltransferases (e.g. N-acetylglucosaminyltransferases, oligosaccharyltransferases, sialyltransferases), glycoaminidases (e.g., PNGase F) and glycosidases. The glycosidases are further classified as exoglycosidases (e.g., p-mannosidase, p-glucosidase), and endoglycosidases (e.g., Endo-A, Endo-M). Each of these classes of enzymes has been successfully used synthetically to prepare carbohydrates and glycoproteins. As an example, RNase B has been synthesized as a high-mannose glycosylated protein, after which the oligosaccharide was enzymatically removed (apart from a single GlcNAc) and the correct glycoform was produced in subsequent transglycosylation reactions using different enzymes (Witte et al., J. Am. Chem. Soc., 119 (9), 2114-2118, 1997). More examples of how transglycosylation may be used in glycoprotein synthesis are reviewed and described in Crout et al., Curr. Opin. Chem. Biol. 2: 98-111 (1998); Arsequell, Tetrahedron: Asymmetry 10: 3045 (1999); Murata et al., 1059 (1997), Murata et al., 1049 (2006), WO2003/046150, WO2007/133855, Koeller et al., Nature Biotechnology 18: 835-841 (2000). However, for efficient transglycosylation by enzymes, a starting population having a uniform glycosylation profile is still highly desirable (cf. e.g. the single GlcNAc population used by Witte et al., J. Am. Chem. Soc., 119 (9), 2114-2118, 1997).
A special situation presents itself in crystallization studies of glycoproteins. Here, N-glycosylation often poses a problem. Indeed, when attempting to crystallize a glycoprotein, the results can be improved when using de-N-glycosylated forms of the target protein. However, mutation of the glycosylation-site is mostly not an option, since N-glycosylation is needed for protein folding and quality-control. At present endoH-type endoglycosidases are often used for the post-purification deglycosylation of high-mannose type glycoproteins. This approach is successful in many cases but contributes to the complexity of the downstream processing of these often labile proteins. Therefore, it would be advantageous to be able to eliminate downstream processing steps and still obtain a population that can be used for crystallization purposes. A similar situation is observed in glycoproteins that are produced in cells which modify them with immunogenic glycans.
Despite the many advantages of the enzymatic synthesis methods set forth above, in some cases, deficiencies remain. The preparation of properly glycosylated glycopeptides is an exemplary situation in which additional effort is required and effort is being directed to improving both the synthesis of glycopeptides and methods of remodeling biologically or chemically produced glycopeptides that are not properly glycosylated. Thus, there is a need to have a cell system or synthesis method providing homogeneous (uniform) glycosylation on a population of glycoproteins, either already with a correct glycoprofile or as a starting point for subsequent transglycosylation. Alternatively, it would be advantageous to have a cell system or synthesis method providing the possibility of easier isolation of the correctly modified population of glycoproteins from a mixed population of glycoproteins. Particularly also for yeast, it would be advantageous to be able to eliminate downstream processing steps, while still being able to easily separate the desired (complex type) glycoproteins from the undesired, possibly immunogenic glycoforms; or even to obtain yeast cells that no longer produce immunogenic glycoproteins.